T0070907

Human aortic smooth muscle cells (HASMCs) from ScienCell Research Laboratories were isolated from human aorta and cultured in smooth muscle cell medium (SMCM, Cat. #1101, ScienCell, USA) supplemented with 2% FBS, 1% SMCGS, and 1% penicillin and streptomycin. Experiments were performed using cells from passages 3–6. For reproducibility, we purchased and applied two batches of HASMCs in the following experiments. HEK293T cells were purchased from China Center for Type Culture Collection (Wuhan University, Hubei, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin and maintained at 37 °C in 5% CO₂. The duplex siRNA targeting NLRC5 (SASI_Hs02_00359503) and scramble siRNA (siCtr) were purchased from Sigma incorporation (Sigma, USA). The siRNA-targeting PPARγ was purchased from Sangon Biotech (Shanghai, China) and the sequences are as followed: sense- 5′-CUG GCC UCC UUG AUG AAU ATT-3′, antisense-5′-UAU UCA UCA AGG AGG CCA GTT-3′. HASMCs, seeded in a six-well plate at the density of 1.5 × 10⁶ cells/well, were transfected with 50 nM siRNA using 3 μl of RNAiMAX (Thermo Fisher, USA) in OPTI-MEM (Thermo Fisher, USA) for 24 h. Pioglitazone (CDS021593, Sigma), a therapeutic drug for diabetes, was used as an agonist of PPARγ. T0070907 (S2871, Selleck Chemicals, USA) was used as an antagonist of PPARγ.

Fluvastatin capsules were purchased from Novartis Pharmaceutical Co., Ltd. (Beijing, China). A Masson staining kit was purchased from Leagene Biotechnology Co., Ltd. (Beijing, China). A hematoxylin-eosin staining kit was purchased from Servicebio Technology Co., Ltd. (Wuhan, China). Cell Counting Kit-8 (CCK-8) was obtained from Fcmacs Biotech Co., Ltd. (Nanjing, China). A BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Rosiglitazone (RSG, selective PPAR-γ agonist) and T0070907 (selective PPAR-γ antagonist) were obtained from Selleck Chemicals (Houston, USA). LPS was purchased from Sigma Chemical (St. Louis, USA). Recombinant rat IL-4 and INF-γ were purchased from PeproTech Inc. (New Jersey, USA). ELISA kits (IL-1, IL-6, TNF-α, IL4, IL-10, IL-13, and TGF-β1) were obtained from JinYiBai Biological Technology (Nanjing, China). The antibodies (JAK2, STAT6, and p-STAT1) were purchased from Santa Cruz Biotechnology (California, USA). The antibodies (PPAR-γ, STAT1, and p-PPAR-γ) were purchased from Bioss (Beijing, China). The antibodies (SOCS1, SOCS3, p-JAK2, and p-STAT6) were purchased from Affinity Biosciences. APC-anti-mouse CD86 was purchased from BioLegend (San Diego, USA). PE-Cy7-anti-mouse CD206 and Intracellular Fixation & Permeabilization set were purchased from Thermo Fisher Scientific (Massachusetts, USA).